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1.
Cell ; 185(7): 1157-1171.e22, 2022 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-35259335

RESUMO

Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.


Assuntos
Toxinas Bacterianas/metabolismo , Enterococcus , Leucócitos Mononucleares , Fatores de Virulência/metabolismo , Animais , Bovinos , Enterococcus/metabolismo , Enterococcus/patogenicidade , Cavalos , Camundongos , Testes de Sensibilidade Microbiana , Suínos
2.
Cardiovasc Diabetol ; 21(1): 252, 2022 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-36397150

RESUMO

BACKGROUND AND AIMS: Although type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD) share many common pathological and physiological characteristics, there are few studies assessing the predictive capacity of novel biomarkers in occurrence and development of CAD in T2DM patients aged ≥ 65 years. In addition, T2DM patients aged ≥ 65 years are prone to CAD. Therefore, it is of great significance to find novel biomarkers for the development CAD in T2DM. METHODS: In this retrospective cohort study, 579 T2DM patients aged ≥ 65 years were consecutively enrolled in this work, and 177 of whom had major adverse cardiovascular and cerebrovascular events (MACCE: cardiovascular or cerebrovascular death, acute coronary syndrome, coronary stent implantation, and stroke) during the follow up. Univariate and multivariate factors were employed to analyze the correlation between each variable and the occurrence of MACCE, and the Spearman's rank correlation analysis was performed to assess the relationships between Neutrophil gelatinase-associated lipocalin (NGAL) and small dense low-density lipoprotein-cholesterol (LDL-C) (sdLDL-C). The receiver operating characteristic (ROC) curve was adopted to determine the predictive value of NGAL and sdLDL-C elevation for MACCE in T2DM patients aged ≥ 65 years. RESULTS: After a median 48 months follow-up [19, (10 ~ 32) ], the levels of NGAL, sdLDL-C, hemoglobin A1c (HbA1c), LDL-C, and apolipoprotein B (ApoB) were significantly higher while those of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A I (ApoA-I) were lower in MACCE positive group. NGAL correlated to body mass index (BMI) (r = 0.391, P = 0.001) and triglyceride (TG) (r = 0.228, P = 0.032), and high-sensitivity CRP (hsCRP) (r = 0.251, P = 0.007), and neutrophils (r = 0.454, P = 0.001), sdlDL-C level was found to be positively correlated with LDL-C (r = 0.413, P = 0.001), TG (r = 0.432, P = 0.001), and ApoB (r = 0.232, P = 0.002); and it was negatively correlated with HDL-C (r = -0.362, P = 0.031) and ApoA-I (r = -0.402, P = 0.001). Age-adjusted Cox regression analysis showed that NGAL (HR = 1.006, 95% confidence interval (CI): 1.005-1.008, P < 0.001) and sdLDL-C (HR = 1.052, 95% CI: 1.037-1.066, P < 0.001) were independently associated with occurrence of MACCE. ROC curve analysis showed that NGAL (area under ROC (AUC) = 0.79, 95% CI: 0.75-0.84, P < 0.001) and sdlDL-C (AUC = 0.76, 95% CI: 0.72-0.80, P < 0.001) could predict the occurrence of MACCE (area under ROC. NGAL combined with sdlDL-C could predict the occurrence of MACCE well (AUC = 0.87, 95% CI: 0.84-0.90, P < 0.001). CONCLUSION: The higher NGAL and sdLDL-C in T2DM patients aged ≥ 65 years were significantly and independently associated with the risk of MACCE, and showed higher clinical values than other lipid biomarkers or other chronic inflammation, so they were expected to be the most effective predictors of MACCE assessment.


Assuntos
Doença da Artéria Coronariana , Diabetes Mellitus Tipo 2 , Humanos , Apolipoproteína A-I , Apolipoproteínas B , Biomarcadores , LDL-Colesterol , Doença da Artéria Coronariana/diagnóstico , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/epidemiologia , Lipocalina-2 , Estudos Retrospectivos , Triglicerídeos , Idoso
3.
Circulation ; 142(20): 1937-1955, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-32929985

RESUMO

BACKGROUND: Calmodulin mutations are associated with arrhythmia syndromes in humans. Exome sequencing previously identified a de novo mutation in CALM1 resulting in a p.N98S substitution in a patient with sinus bradycardia and stress-induced bidirectional ventricular ectopy. The objectives of the present study were to determine if mice carrying the N98S mutation knocked into Calm1 replicate the human arrhythmia phenotype and to examine arrhythmia mechanisms. METHODS: Mouse lines heterozygous for the Calm1N98S allele (Calm1N98S/+) were generated using CRISPR/Cas9 technology. Adult mutant mice and their wildtype littermates (Calm1+/+) underwent electrocardiographic monitoring. Ventricular de- and repolarization was assessed in isolated hearts using optical voltage mapping. Action potentials and whole-cell currents and [Ca2+]i, as well, were measured in single ventricular myocytes using the patch-clamp technique and fluorescence microscopy, respectively. The microelectrode technique was used for in situ membrane voltage monitoring of ventricular conduction fibers. RESULTS: Two biologically independent knock-in mouse lines heterozygous for the Calm1N98S allele were generated. Calm1N98S/+ mice of either sex and line exhibited sinus bradycardia, QTc interval prolongation, and catecholaminergic bidirectional ventricular tachycardia. Male mutant mice also showed QRS widening. Pharmacological blockade and activation of ß-adrenergic receptors rescued and exacerbated, respectively, the long-QT phenotype of Calm1N98S/+ mice. Optical and electric assessment of membrane potential in isolated hearts and single left ventricular myocytes, respectively, revealed ß-adrenergically induced delay of repolarization. ß-Adrenergic stimulation increased peak density, slowed inactivation, and left-shifted the activation curve of ICa.L significantly more in Calm1N98S/+ versus Calm1+/+ ventricular myocytes, increasing late ICa.L in the former. Rapidly paced Calm1N98S/+ ventricular myocytes showed increased propensity to delayed afterdepolarization-induced triggered activity, whereas in situ His-Purkinje fibers exhibited increased susceptibility for pause-dependent early afterdepolarizations. Epicardial mapping of Calm1N98S/+ hearts showed that both reentry and focal mechanisms contribute to arrhythmogenesis. CONCLUSIONS: Heterozygosity for the Calm1N98S mutation is causative of an arrhythmia syndrome characterized by sinus bradycardia, QRS widening, adrenergically mediated QTc interval prolongation, and bidirectional ventricular tachycardia. ß-Adrenergically induced ICa.L dysregulation contributes to the long-QT phenotype. Pause-dependent early afterdepolarizations and tachycardia-induced delayed afterdepolarizations originating in the His-Purkinje network and ventricular myocytes, respectively, constitute potential sources of arrhythmia in Calm1N98S/+ hearts.


Assuntos
Calmodulina , Ventrículos do Coração/metabolismo , Mutação de Sentido Incorreto , Miócitos Cardíacos/metabolismo , Ramos Subendocárdicos/metabolismo , Síndrome do Nó Sinusal/congênito , Substituição de Aminoácidos , Animais , Calmodulina/genética , Calmodulina/metabolismo , Modelos Animais de Doenças , Ventrículos do Coração/fisiopatologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Ramos Subendocárdicos/fisiopatologia , Síndrome do Nó Sinusal/genética , Síndrome do Nó Sinusal/metabolismo , Síndrome do Nó Sinusal/fisiopatologia
4.
Circulation ; 141(23): 1915-1926, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32511001

RESUMO

The interrelationships between atrial fibrillation (AF) and heart failure (HF) are complex and poorly understood, yet the number of patients with AF and HF continues to increase worldwide. Thus, there is a need for initiatives that prioritize research on the intersection between AF and HF. This article summarizes the proceedings of a virtual workshop convened by the US National Heart, Lung, and Blood Institute to identify important research opportunities in AF and HF. Key knowledge gaps were reviewed and research priorities were proposed for characterizing the pathophysiological overlap and deleterious interactions between AF and HF; preventing HF in people with AF; preventing AF in individuals with HF; and addressing symptom burden and health status outcomes in AF and HF. These research priorities will hopefully help inform, encourage, and stimulate innovative, cost-efficient, and transformative studies to enhance the outcomes of patients with AF and HF.


Assuntos
Fibrilação Atrial/epidemiologia , Pesquisa Biomédica/normas , Educação/normas , Insuficiência Cardíaca/epidemiologia , National Heart, Lung, and Blood Institute (U.S.)/normas , Relatório de Pesquisa/normas , Fibrilação Atrial/terapia , Educação/métodos , Insuficiência Cardíaca/terapia , Humanos , Estudos Observacionais como Assunto/métodos , Estudos Observacionais como Assunto/normas , Estados Unidos/epidemiologia
5.
Pflugers Arch ; 473(3): 491-506, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33411079

RESUMO

Apamin-sensitive small-conductance calcium-activated potassium (SK) current (IKAS) plays an important role in cardiac repolarization under a variety of physiological and pathological conditions. The regulation of cardiac IKAS relies on SK channel expression, intracellular Ca2+, and interaction between SK channel and intracellular Ca2+. IKAS activation participates in multiple types of arrhythmias, including atrial fibrillation, ventricular tachyarrhythmias, and automaticity and conduction abnormality. Recently, sex dimorphisms in autonomic control have been noticed in IKAS activation, resulting in sex-differentiated action potential morphology and arrhythmogenesis. This review provides an update on the Ca2+-dependent regulation of cardiac IKAS and the role of IKAS on arrhythmias, with a special focus on sex differences in IKAS activation. We propose that sex dimorphism in autonomic control of IKAS may play a role in J wave syndrome.


Assuntos
Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Caracteres Sexuais , Canais de Potássio Ativados por Cálcio de Condutância Baixa/fisiologia , Animais , Feminino , Humanos , Masculino
6.
Am J Physiol Heart Circ Physiol ; 320(2): H826-H837, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33385322

RESUMO

Spiral wave reentry as a mechanism of lethal ventricular arrhythmias has been widely demonstrated in animal experiments and recordings from human hearts. It has been shown that in structurally normal hearts spiral waves are unstable, breaking up into multiple wavelets via dynamical instabilities. However, many of the second-generation action potential models give rise only to stable spiral waves, raising issues regarding the underlying mechanisms of spiral wave breakup. In this study, we carried out computer simulations of two-dimensional homogeneous tissues using five ventricular action potential models. We show that the transient outward potassium current (Ito), although it is not required, plays a key role in promoting spiral wave breakup in all five models. As the maximum conductance of Ito increases, it first promotes spiral wave breakup and then stabilizes the spiral waves. In the absence of Ito, speeding up the L-type calcium kinetics can prevent spiral wave breakup, however, with the same speedup kinetics, spiral wave breakup can be promoted by increasing Ito. Increasing Ito promotes single-cell dynamical instabilities, including action potential duration alternans and chaos, and increasing Ito further suppresses these action potential dynamics. These cellular properties agree with the observation that increasing Ito first promotes spiral wave breakup and then stabilizes spiral waves in tissue. Implications of our observations to spiral wave dynamics in the real hearts and action potential model improvements are discussed.NEW & NOTEWORTHY Spiral wave breakup manifesting as multiple wavelets is a mechanism of ventricular fibrillation. It has been known that spiral wave breakup in cardiac tissue can be caused by a steeply sloped action potential duration restitution curve, a property mainly determined by the recovery of L-type calcium current. Here, we show that the transient outward potassium current (Ito) is another current that plays a key role in spiral wave breakup, that is, spiral waves can be stable for low and high maximum Ito conductance but breakup occurs for intermediate maximum Ito conductance. Since Ito is present in normal hearts of many species and required for Brugada syndrome, it may play an important role in the spiral wave stability and arrhythmogenesis under both normal condition and Brugada syndrome.


Assuntos
Potenciais de Ação , Arritmias Cardíacas/etiologia , Frequência Cardíaca , Ventrículos do Coração/metabolismo , Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Canais de Potássio/metabolismo , Potássio/metabolismo , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Simulação por Computador , Cobaias , Ventrículos do Coração/fisiopatologia , Humanos , Cinética , Coelhos
7.
Circ Res ; 125(6): 575-589, 2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366290

RESUMO

RATIONALE: The ventricular conduction system (VCS) rapidly propagates electrical impulses through the working myocardium of the ventricles to coordinate chamber contraction. GWAS (Genome-wide association studies) have associated nucleotide polymorphisms, most are located within regulatory intergenic or intronic sequences, with variation in VCS function. Two highly correlated polymorphisms (r2>0.99) associated with VCS functional variation (rs13165478 and rs13185595) occur 5' to the gene encoding the basic helix-loop-helix transcription factor HAND1 (heart- and neural crest derivatives-expressed protein 1). OBJECTIVE: Here, we test the hypothesis that these polymorphisms influence HAND1 transcription thereby influencing VCS development and function. METHODS AND RESULTS: We employed transgenic mouse models to identify an enhancer that is sufficient for left ventricle (LV) cis-regulatory activity. Two evolutionarily conserved GATA transcription factor cis-binding elements within this enhancer are bound by GATA4 and are necessary for cis-regulatory activity, as shown by in vitro DNA binding assays. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated deletion of this enhancer dramatically reduces Hand1 expression solely within the LV but does not phenocopy previously published mouse models of cardiac Hand1 loss-of-function. Electrophysiological and morphological analyses reveals that mice homozygous for this deleted enhancer display a morphologically abnormal VCS and a conduction system phenotype consistent with right bundle branch block. Using 1000 Genomes Project data, we identify 3 additional single nucleotide polymorphisms (SNPs), located within the Hand1 LV enhancer, that compose a haplotype with rs13165478 and rs13185595. One of these SNPs, rs10054375, overlaps with a critical GATA cis-regulatory element within the Hand1 LV enhancer. This SNP, when tested in electrophoretic mobility shift assays, disrupts GATA4 DNA-binding. Modeling 2 of these SNPs in mice causes diminished Hand1 expression and mice present with abnormal VCS function. CONCLUSIONS: Together, these findings reveal that SNP rs10054375, which is located within a necessary and sufficient LV-specific Hand1 enhancer, exhibits reduces GATA DNA-binding in electrophoretic mobility shift assay, and this enhancer in total, is required for VCS development and function in mice and perhaps humans.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Desenvolvimento Embrionário/fisiologia , Fator de Transcrição GATA4/metabolismo , Variação Genética/fisiologia , Sistema de Condução Cardíaco/fisiologia , Função Ventricular/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Feminino , Fator de Transcrição GATA4/genética , Ventrículos do Coração/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Polimorfismo de Nucleotídeo Único/fisiologia , Ligação Proteica/fisiologia , Distribuição Aleatória , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Mol Cell Proteomics ; 18(7): 1320-1329, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30992312

RESUMO

BRCA1-associated protein 1 (BAP1) is a member of the ubiquitin C-terminal hydrolase family of deubiquitinating enzymes and is implicated in transcriptional regulation. The BAP1 gene is mutated in about 10% of patients with ccRCC, the most common form of renal cancer, suggesting that BAP1 is a tumor suppressor. However, whether BAP1 influences the progression of ccRCC tumors expressing wild-type (WT) BAP1 is unclear. Here, we assessed the expression and function of BAP1 using human ccRCC specimens and cell lines. Analysis of datasets in The Cancer Genome Atlas revealed that lower BAP1 expression is correlated with longer overall survival of ccRCC patients. We established human ccRCC cell lines with stable BAP1 knockout and performed multiomic analysis of BAP1-mediated cellular processes. BAP1 knockout downregulated proteins associated with protein synthesis, resulting in decreased cell growth. Importantly, loss of BAP1 decreased the formation of stress fibers and membrane protrusions and induced migration and invasion defects. BAP1 knockout in ccRCC cells also downregulated the expression of transcriptional repressor protein Snail and decreased the activity of Rho family GTPases, promoting the cells to undergo mesenchymal-epithelial transition. Unexpectedly, quantitative proteomics also showed that BAP1 knockout increased expression of several amino acid transporters and multiple tyrosine kinases, including the epidermal growth factor receptor. Overall, our results suggest that BAP1 regulates multiple cellular processes, and we also uncover a new role for BAP1 in controlling mesenchymal-epithelial transition in ccRCC cells.


Assuntos
Transição Epitelial-Mesenquimal , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Proteínas Supressoras de Tumor/deficiência , Ubiquitina Tiolesterase/deficiência , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Humanos , Neoplasias Renais/genética , Proteoma/metabolismo , Proteômica , Fatores de Transcrição da Família Snail/metabolismo , Fibras de Estresse/metabolismo , Análise de Sobrevida , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação
9.
Pacing Clin Electrophysiol ; 43(8): 838-846, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32588437

RESUMO

BACKGROUND: Telethonin (TCAP) is a Z-disk protein that maintains cytoskeletal integrity and various signaling pathways in cardiomyocytes. TCAP is shown to modulate α-subunit of the human cardiac sodium channel (hNav 1.5) by direct interactions. Several TCAP variants are found in cardiomyopathies. We sought to investigate whether TCAP variants are associated with arrhythmia syndromes. METHODS: Mutational analyses for TCAP were performed in 303 Japanese patients with Brugada syndrome, arrhythmogenic right ventricular cardiomyopathy, and J-wave pattern ECG. Using patch-clamp techniques, electrophysiological characteristics of hNav 1.5 were studied in HEK-293 cells stably expressing hNav 1.5 and transiently transfected with wild-type (WT) or variant TCAP. RESULTS: We identified two TCAP variants, c.145G>A:p.E49K and c.458G>A:p.R153H, in four individuals. p.E49K was found in two patients with ARVC or BrS. p.R153H was found in two patients with BrS or J-wave pattern ECG. No patient had variant hNav 1.5. Patch-clamp experiments demonstrated that peak sodium currents were significantly reduced in cells expressing p.R153H and p.E49K compared with WT-TCAP (66%, p.R153H; 72%, p.E49K). Voltage dependency of peak IV curve was rightward-shifted by 5 mV in cells expressing p.E49K compared with WT-TCAP. Voltage dependency of activation was not leftward-shifted by p.R153H, while voltage dependency of steady-state inactivation was leftward-shifted by p.E49K. CONCLUSIONS: We found two TCAP variants in the patients with BrS, J-wave pattern ECG, and ARVC that can cause loss-of-function of the hNav 1.5 in heterologous expression systems. Our observation suggests that these variants might impair INa and be associated with the patients' electrophysiological phenotypes. Further studies linking our experimental data to clinical phenotypes are warranted.


Assuntos
Displasia Arritmogênica Ventricular Direita/genética , Síndrome de Brugada/genética , Conectina/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Adolescente , Adulto , Idoso , Displasia Arritmogênica Ventricular Direita/fisiopatologia , Síndrome de Brugada/fisiopatologia , Eletrocardiografia , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Patch-Clamp
10.
Nature ; 514(7520): 102-106, 2014 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-25119045

RESUMO

The role of long noncoding RNA (lncRNA) in adult hearts is unknown; also unclear is how lncRNA modulates nucleosome remodelling. An estimated 70% of mouse genes undergo antisense transcription, including myosin heavy chain 7 (Myh7), which encodes molecular motor proteins for heart contraction. Here we identify a cluster of lncRNA transcripts from Myh7 loci and demonstrate a new lncRNA-chromatin mechanism for heart failure. In mice, these transcripts, which we named myosin heavy-chain-associated RNA transcripts (Myheart, or Mhrt), are cardiac-specific and abundant in adult hearts. Pathological stress activates the Brg1-Hdac-Parp chromatin repressor complex to inhibit Mhrt transcription in the heart. Such stress-induced Mhrt repression is essential for cardiomyopathy to develop: restoring Mhrt to the pre-stress level protects the heart from hypertrophy and failure. Mhrt antagonizes the function of Brg1, a chromatin-remodelling factor that is activated by stress to trigger aberrant gene expression and cardiac myopathy. Mhrt prevents Brg1 from recognizing its genomic DNA targets, thus inhibiting chromatin targeting and gene regulation by Brg1. It does so by binding to the helicase domain of Brg1, a domain that is crucial for tethering Brg1 to chromatinized DNA targets. Brg1 helicase has dual nucleic-acid-binding specificities: it is capable of binding lncRNA (Mhrt) and chromatinized--but not naked--DNA. This dual-binding feature of helicase enables a competitive inhibition mechanism by which Mhrt sequesters Brg1 from its genomic DNA targets to prevent chromatin remodelling. A Mhrt-Brg1 feedback circuit is thus crucial for heart function. Human MHRT also originates from MYH7 loci and is repressed in various types of myopathic hearts, suggesting a conserved lncRNA mechanism in human cardiomyopathy. Our studies identify a cardioprotective lncRNA, define a new targeting mechanism for ATP-dependent chromatin-remodelling factors, and establish a new paradigm for lncRNA-chromatin interaction.


Assuntos
Cardiomegalia/genética , Cardiomegalia/patologia , Cadeias Pesadas de Miosina/genética , RNA Longo não Codificante/genética , Animais , Miosinas Cardíacas/genética , Cardiomegalia/prevenção & controle , Cardiomiopatias/genética , Cardiomiopatias/patologia , Cardiomiopatias/prevenção & controle , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA Helicases/antagonistas & inibidores , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/metabolismo , Retroalimentação Fisiológica , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/prevenção & controle , Histona Desacetilases/metabolismo , Humanos , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
Am J Physiol Heart Circ Physiol ; 316(2): H371-H379, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30499712

RESUMO

Cardiomyocyte-restricted overexpression of FK506-binding protein 12 transgenic (αMyHC-FKBP12) mice develop spontaneous atrial fibrillation (AF). The aim of the present study is to explore the mechanisms underlying the occurrence of AF in αMyHC-FKBP12 mice. Spontaneous AF was documented by telemetry in vivo and Langendorff-perfused hearts of αMyHC-FKBP12 and littermate control mice in vitro. Atrial conduction velocity was evaluated by optical mapping. The patch-clamp technique was applied to determine the potentially altered electrophysiology in atrial myocytes. Channel protein expression levels were evaluated by Western blot analyses. Spontaneous AF was recorded in four of seven αMyHC-FKBP12 mice but in none of eight nontransgenic (NTG) controls. Atrial conduction velocity was significantly reduced in αMyHC-FKBP12 hearts compared with NTG hearts. Interestingly, the mean action potential duration at 50% but not 90% was significantly prolonged in αMyHC-FKBP12 atrial myocytes compared with their NTG counterparts. Consistent with decreased conduction velocity, average peak Na+ current ( INa) density was dramatically reduced and the INa inactivation curve was shifted by approximately +7 mV in αMyHC-FKBP12 atrial myocytes, whereas the activation and recovery curves were unaltered. The Nav1.5 expression level was significantly reduced in αMyHC-FKBP12 atria. Furthermore, we found increases in atrial Cav1.2 protein levels and peak L-type Ca2+ current density and increased levels of fibrosis in αMyHC-FKBP12 atria. In summary, cardiomyocyte-restricted overexpression of FKBP12 reduces the atrial Nav1.5 expression level and mean peak INa, which is associated with increased peak L-type Ca2+ current and interstitial fibrosis in atria. The combined electrophysiological and structural changes facilitated the development of local conduction block and altered action potential duration and spontaneous AF. NEW & NOTEWORTHY This study addresses a long-standing riddle regarding the role of FK506-binding protein 12 in cardiac physiology. The work provides further evidence that FK506-binding protein 12 is a critical component for regulating voltage-gated sodium current and in so doing has an important role in arrhythmogenic physiology, such as atrial fibrillation.


Assuntos
Fibrilação Atrial/genética , Proteína 1A de Ligação a Tacrolimo/metabolismo , Potenciais de Ação , Animais , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Proteína 1A de Ligação a Tacrolimo/genética
13.
J Mol Cell Cardiol ; 123: 185-197, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30261161

RESUMO

AIMS: Phospholamban (PLB) is the key regulator of the cardiac Ca2+ pump (SERCA2a)-mediated sarcoplasmic reticulum Ca2+ stores. We recently reported that PLB is highly concentrated in the nuclear envelope (NE) from where it can modulate perinuclear Ca2+ handling of the cardiomyocytes (CMs). Since inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) mediates nuclear Ca2+ release, we examined whether the nuclear pool of PLB regulates IP3-induced nuclear Ca2+ handling. METHODS AND RESULTS: Fluo-4 based confocal Ca2+ imaging was performed to measure Ca2+ dynamics across both nucleus and cytosol in saponin-permeabilized CMs isolated from wild-type (WT) or PLB-knockout (PLB-KO) mice. At diastolic intracellular Ca2+ ([Ca2+]i = 100 nM), the Fab fragment of the monoclonal PLB antibody (anti-PLB Fab) facilitated the formation and increased the length of spontaneous Ca2+ waves (SCWs) originating from the nuclear region in CMs from WT but not from PLB-KO mice. We next examined nuclear Ca2+ activities at basal condition and after sequential addition of IP3, anti-PLB Fab, and the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) at a series of [Ca2+]i. In WT mice, at 10 nM [Ca2+]i where ryanodine receptor (RyR2) based spontaneous Ca2+ sparks rarely occurred, IP3 increased fluorescence amplitude (F/F0) of overall nuclear region to 1.19 ±â€¯0.02. Subsequent addition of anti-PLB Fab significantly decreased F/F0 to 1.09 ±â€¯0.02. At 50 nM [Ca2+]i, anti-PLB Fab not only decreased the overall nuclear F/F0 previously elevated by IP3, but also increased the amplitude and duration of spark-like nuclear Ca2+ release events. These nuclear Ca2+ releases were blocked by 2-APB. At 100 nM [Ca2+]i, IP3 induced short SCWs originating from nucleus. Anti-PLB Fab transformed those short waves into long SCWs with propagation from the nucleus into the cytosol. In contrast, neither nuclear nor cytosolic Ca2+ dynamics was affected by anti-PLB Fab in CMs from PLB-KO mice in all these conditions. Furthermore, in WT CMs pretreated with RyR2 blocker tetracaine, IP3 and anti-PLB Fab still increased the magnitude of nuclear Ca2+ release but failed to regenerate SCWs. Finally, anti-PLB Fab increased low Ca2+ affinity mag-fluo 4 fluorescence intensity in the lumen of NE of nuclei isolated from WT but not in PLB-KO mice. CONCLUSION: PLB regulates nuclear Ca2+ handling. By increasing Ca2+ uptake into lumen of the NE and perhaps other perinuclear membranes, the acute reversal of PLB inhibition decreases global Ca2+ concentration at rest in the nucleoplasm, and increases Ca2+ release into the nucleus, through mechanisms involving IP3R and RyR2 in the vicinity.


Assuntos
Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Núcleo Celular/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Cães , Camundongos , Camundongos Knockout , Imagem Molecular/métodos , Miócitos Cardíacos/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Tetracaína/farmacologia
14.
J Physiol ; 596(18): 4299-4322, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29917243

RESUMO

KEY POINTS: It is unknown if a sex difference exists in cardiac apamin-sensitive small conductance Ca2+ -activated K+ (SK) current (IKAS ). There is no sex difference in IKAS in the basal condition. However, there is larger IKAS in female rabbit ventricles than in male during isoproterenol infusion. IKAS activation by isoproterenol leads to action potential triangulation in females, indicating its abundant activation at early phases of repolarization. IKAS activation in females induces negative Ca2+ -voltage coupling and promotes electromechanically discordant phase 2 repolarization alternans. IKAS is important in the mechanisms of ventricular fibrillation in females during sympathetic stimulation. ABSTRACT: Sex has a large influence on cardiac electrophysiological properties. Whether sex differences exist in apamin-sensitive small conductance Ca2+ -activated K+ (SK) current (IKAS ) remains unknown. We performed optical mapping, transmembrane potential, patch clamp, western blot and immunostaining in 62 normal rabbit ventricles, including 32 females and 30 males. IKAS blockade by apamin only minimally prolonged action potential (AP) duration (APD) in the basal condition for both sexes, but significantly prolonged APD in the presence of isoproterenol in females. Apamin prolonged APD at the level of 25% repolarization (APD25 ) more prominently than APD at the level of 80% repolarization (APD80 ), consequently reversing isoproterenol-induced AP triangulation in females. In comparison, apamin prolonged APD to a significantly lesser extent in males and failed to restore the AP plateau during isoproterenol infusion. IKAS in males did not respond to the L-type calcium current agonist BayK8644, but was amplified by the casein kinase 2 (CK2) inhibitor 4,5,6,7-tetrabromobenzotriazole. In addition, whole-cell outward IKAS densities in ventricular cardiomyocytes were significantly larger in females than in males. SK channel subtype 2 (SK2) protein expression was higher and the CK2/SK2 ratio was lower in females than in males. IKAS activation in females induced negative intracellular Ca2+ -voltage coupling, promoted electromechanically discordant phase 2 repolarization alternans and facilitated ventricular fibrillation (VF). Apamin eliminated the negative Ca2+ -voltage coupling, attenuated alternans and reduced VF inducibility, phase singularities and dominant frequencies in females, but not in males. We conclude that ß-adrenergic stimulation activates ventricular IKAS in females to a much greater extent than in males. IKAS activation plays an important role in ventricular arrhythmogenesis in females during sympathetic stimulation.


Assuntos
Potenciais de Ação , Agonistas Adrenérgicos beta/farmacologia , Frequência Cardíaca , Ventrículos do Coração/metabolismo , Isoproterenol/farmacologia , Miócitos Cardíacos/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Animais , Apamina/farmacologia , Células Cultivadas , Feminino , Ventrículos do Coração/efeitos dos fármacos , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Coelhos , Fatores Sexuais , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores
15.
Am J Physiol Heart Circ Physiol ; 315(2): H375-H388, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29677462

RESUMO

Apamin-sensitive small-conductance Ca2+-activated K+ (SK) current ( IKAS) is encoded by Ca2+-activated K+ channel subfamily N ( KCNN) genes. IKAS importantly contributes to cardiac repolarization in conditions associated with reduced repolarization reserve. To test the hypothesis that IKAS inhibition contributes to drug-induced long QT syndrome (diLQTS), we screened for KCNN variants among patients with diLQTS, determined the properties of heterologously expressed wild-type (WT) and variant KCNN channels, and determined if the 5-HT3 receptor antagonist ondansetron blocks IKAS. We searched 2,306,335 records in the Indiana Network for Patient Care and found 11 patients with diLQTS who had DNA available in the Indiana Biobank. DNA sequencing discovered a heterozygous KCNN2 variant (p.F503L) in a 52-yr-old woman presenting with corrected QT interval prolongation at baseline (473 ms) and further corrected QT interval lengthening (601 ms) after oral administration of ondansetron. That patient was also heterozygous for the p.S38G and p.P2835S variants of the QT-controlling genes KCNE1 and ankyrin 2, respectively. Patch-clamp experiments revealed that the p.F503L KCNN2 variant heterologously expressed in human embryonic kidney (HEK)-293 cells augmented Ca2+ sensitivity, increasing IKAS density. The fraction of total F503L-KCNN2 protein retained in the membrane was higher than that of WT KCNN2 protein. Ondansetron at nanomolar concentrations inhibited WT and p.F503L SK2 channels expressed in HEK-293 cells as well as native SK channels in ventricular cardiomyocytes. Ondansetron-induced IKAS inhibition was also demonstrated in Langendorff-perfused murine hearts. In conclusion, the heterozygous p.F503L KCNN2 variant increases Ca2+ sensitivity and IKAS density in transfected HEK-293 cells. Ondansetron at therapeutic (i.e., nanomolar) concentrations is a potent IKAS blocker. NEW & NOTEWORTHY We showed that ondansetron, a 5-HT3 receptor antagonist, blocks small-conductance Ca2+-activated K+ (SK) current. Ondansetron may be useful in controlling arrhythmias in which increased SK current is a likely contributor. However, its SK-blocking effects may also facilitate the development of drug-induced long QT syndrome.


Assuntos
Antiarrítmicos/farmacologia , Síndrome do QT Longo/tratamento farmacológico , Ondansetron/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Animais , Antiarrítmicos/uso terapêutico , Cálcio/metabolismo , Células Cultivadas , Feminino , Células HEK293 , Humanos , Síndrome do QT Longo/genética , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Ondansetron/uso terapêutico , Bloqueadores dos Canais de Potássio/uso terapêutico , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo
16.
J Cardiovasc Electrophysiol ; 28(12): 1485-1491, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28833764

RESUMO

The autonomic nervous system plays an important role in the genesis of atrial fibrillation and is one of the candidate targets for atrial fibrillation therapy. This review focuses on the role of the autonomic nervous system in atrial fibrillation development and discusses the results of the ganglionated plexi catheter and surgical ablation in preclinical and clinical studies. The heart is innervated by the extrinsic and intrinsic autonomic nervous systems. The intrinsic autonomic nervous system consists of multiple ganglionated plexi and axons, which innervate the neighboring atrial myocardium and control their electrophysiological properties. Abnormal autonomic innervation has been observed in an animal model of atrial fibrillation and in humans. Direct recordings of autonomic nerve activity in canine models showed that atrial tachyarrhythmia episodes were invariably preceded by intrinsic cardiac autonomic nerve activity, thus supporting the importance of intrinsic cardiac autonomic nerve activity as the triggers for atrial tachyarrhythmia. Targeting ganglionated plexi with catheter ablation improves the outcomes of paroxysmal atrial fibrillation ablation in addition to pulmonary vein antrum isolation. Ablation of ganglionated plexi alone without pulmonary vein isolation is also useful in controlling paroxysmal atrial fibrillation in some patients. However, surgical ganglionated plexi ablation in patients with a large left atrium, persistent atrial fibrillation, and/or a history of prior catheter ablation does not result in additional benefits. These different outcomes suggest that ganglionated plexi ablation is effective in managing patients with paroxysmal atrial fibrillation, but its effects in patients with persistent atrial fibrillation and advanced atrial diseases might be limited.


Assuntos
Fibrilação Atrial/fisiopatologia , Fibrilação Atrial/cirurgia , Ablação por Cateter/métodos , Gânglios Autônomos/fisiopatologia , Animais , Sistema de Condução Cardíaco/fisiopatologia , Sistema de Condução Cardíaco/cirurgia , Humanos , Neurotransmissores/fisiologia
17.
J Mol Cell Cardiol ; 100: 1-8, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27642167

RESUMO

AIMS: Phospholamban (PLB) regulates the cardiac Ca2+-ATPase (SERCA2a) in sarcoplasmic reticulum (SR). However, the localization of PLB at subcellular sites outside the SR and possible contributions to Ca2+ cycling remain unknown. We examined the intracellular distribution of PLB and tested whether a pool of PLB exists in the nuclear envelope (NE) that might regulate perinuclear/nuclear Ca2+ (nCa2+) handling in cardiomyocytes (CMs). METHODS AND RESULTS: Using confocal immunofluorescence microscopy and immunoblot analyses of CMs and CM nuclei, we discovered that PLB was highly concentrated in NE. Moreover, the ratio of PLB levels to SERCA levels was greater in NE than in SR. The increased levels of PLB in NE were a consistent finding using a range of antibodies, tissue samples, and species. To address a possible role in affecting Ca2+ handling, we used Fluo-4 based confocal Ca2+ imaging, with scan-lines across cytosol and nuclei, and evaluated the effects of PLB on cytosolic and nCa2+ uptake and release in mouse CMs. In intact CMs, isoproterenol increased amplitude and decreased the decay time of Ca2+ transients not only in cytosol but also in nuclear regions. In saponin-permeabilized mouse CMs ([Ca2+]i=400nM), we measured spontaneous Ca2+ waves after specific reversal of PLB activity by addition of the Fab fragment of an anti-PLB monoclonal antibody (100µg/ml). This highly selective immunological reagent enhanced Ca2+ uptake (faster decay times) and Ca2+ release (greater intensity) in both cytosol and across the nuclear regions. CONCLUSIONS: Besides SR, PLB is concentrated in NE of CMs, and may be involved in modulation of nCa2+ dynamics.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Membrana Nuclear/metabolismo , Animais , Transporte Biológico , Sinalização do Cálcio/efeitos dos fármacos , Nucléolo Celular/metabolismo , Humanos , Espaço Intracelular/metabolismo , Isoproterenol/farmacologia , Camundongos , Microscopia de Fluorescência , Imagem Molecular , Miócitos Cardíacos/efeitos dos fármacos , Coelhos , Especificidade da Espécie
18.
J Physiol ; 594(14): 3911-54, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27114333

RESUMO

The autonomic nervous system regulates all aspects of normal cardiac function, and is recognized to play a critical role in the pathophysiology of many cardiovascular diseases. As such, the value of neuroscience-based cardiovascular therapeutics is increasingly evident. This White Paper reviews the current state of understanding of human cardiac neuroanatomy, neurophysiology, pathophysiology in specific disease conditions, autonomic testing, risk stratification, and neuromodulatory strategies to mitigate the progression of cardiovascular diseases.


Assuntos
Doenças Cardiovasculares/fisiopatologia , Coração/inervação , Coração/fisiologia , Animais , Sistema Nervoso Autônomo/fisiologia , Doenças Cardiovasculares/terapia , Coração/fisiopatologia , Humanos
19.
Circulation ; 132(15): 1377-86, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26362634

RESUMO

BACKGROUND: Hypokalemia increases the vulnerability to ventricular fibrillation. We hypothesize that the apamin-sensitive small-conductance calcium-activated potassium current (IKAS) is activated during hypokalemia and that IKAS blockade is proarrhythmic. METHODS AND RESULTS: Optical mapping was performed in 23 Langendorff-perfused rabbit ventricles with atrioventricular block and either right or left ventricular pacing during normokalemia or hypokalemia. Apamin prolonged the action potential duration (APD) measured to 80% repolarization (APD80) by 26 milliseconds (95% confidence interval [CI], 14-37) during normokalemia and by 54 milliseconds (95% CI, 40-68) during hypokalemia (P=0.01) at a 1000-millisecond pacing cycle length. In hypokalemic ventricles, apamin increased the maximal slope of APD restitution, the pacing cycle length threshold of APD alternans, the pacing cycle length for wave-break induction, and the area of spatially discordant APD alternans. Apamin significantly facilitated the induction of sustained ventricular fibrillation (from 3 of 9 hearts to 9 of 9 hearts; P=0.009). Short-term cardiac memory was assessed by the slope of APD80 versus activation time. The slope increased from 0.01 (95% CI, -0.09 to 0.12) at baseline to 0.34 (95% CI, 0.23-0.44) after apamin (P<0.001) during right ventricular pacing and from 0.07 (95% CI, -0.05 to 0.20) to 0.54 (95% CI, 0.06-1.03) after apamin infusion (P=0.045) during left ventricular pacing. Patch-clamp studies confirmed increased IKAS in isolated rabbit ventricular myocytes during hypokalemia (P=0.038). CONCLUSIONS: Hypokalemia activates IKAS to shorten APD and maintain repolarization reserve at late activation sites during ventricular pacing. IKAS blockade prominently lengthens the APD at late activation sites and facilitates ventricular fibrillation induction.


Assuntos
Estimulação Cardíaca Artificial , Sistema de Condução Cardíaco/fisiopatologia , Hipopotassemia/fisiopatologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/fisiologia , Potássio/fisiologia , Fibrilação Ventricular/etiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Apamina/farmacologia , Estimulação Cardíaca Artificial/efeitos adversos , Suscetibilidade a Doenças , Sistema de Condução Cardíaco/efeitos dos fármacos , Ventrículos do Coração/fisiopatologia , Hipopotassemia/complicações , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Coelhos , Fibrilação Ventricular/fisiopatologia , Fibrilação Ventricular/prevenção & controle , Imagens com Corantes Sensíveis à Voltagem
20.
Circ Res ; 114(9): 1500-15, 2014 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-24763467

RESUMO

Autonomic nervous system activation can induce significant and heterogeneous changes of atrial electrophysiology and induce atrial tachyarrhythmias, including atrial tachycardia and atrial fibrillation (AF). The importance of the autonomic nervous system in atrial arrhythmogenesis is also supported by circadian variation in the incidence of symptomatic AF in humans. Methods that reduce autonomic innervation or outflow have been shown to reduce the incidence of spontaneous or induced atrial arrhythmias, suggesting that neuromodulation may be helpful in controlling AF. In this review, we focus on the relationship between the autonomic nervous system and the pathophysiology of AF and the potential benefit and limitations of neuromodulation in the management of this arrhythmia. We conclude that autonomic nerve activity plays an important role in the initiation and maintenance of AF, and modulating autonomic nerve function may contribute to AF control. Potential therapeutic applications include ganglionated plexus ablation, renal sympathetic denervation, cervical vagal nerve stimulation, baroreflex stimulation, cutaneous stimulation, novel drug approaches, and biological therapies. Although the role of the autonomic nervous system has long been recognized, new science and new technologies promise exciting prospects for the future.


Assuntos
Antiarrítmicos/uso terapêutico , Fibrilação Atrial/terapia , Função Atrial/efeitos dos fármacos , Denervação Autônoma , Sistema Nervoso Autônomo/efeitos dos fármacos , Sistema Nervoso Autônomo/cirurgia , Ablação por Cateter , Potenciais de Ação , Animais , Fibrilação Atrial/fisiopatologia , Denervação Autônoma/métodos , Sistema Nervoso Autônomo/fisiopatologia , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/inervação , Átrios do Coração/fisiopatologia , Átrios do Coração/cirurgia , Humanos , Fatores de Risco , Resultado do Tratamento
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